Bone formation and repair are complex processes involving alterations in gene expression with a concomitant increase of cells expressing proteins and enzymes essential for angiogenesis, matrix synthesis, and mineralization. Our long range goal is to gain insight into the regulation of osteogenic pathways under physiological conditions in living, intact bone. The hypothesis underlying this proposal is that due to dynamic and milieu-dependent regulation, many molecular bone formation pathways are concealed or different in vitro or even in situ as compared to in vivo. In an innovative approach, we will combine molecular imaging in vivo with conventional post mortem analysis. Imaging will offer non-invasive observation of bone biology under physiological conditions, while ex vivo analysis will allow for validation of the in vivo findings and for correlation between in vivo and in vitro analyses. Given that transcriptional programs initiate and govern osteogenesis, the first specific aim is directed to address our incomplete understanding of how angiogenic gene expression is regulated during bone healing. Utilizing innovative transgenic imaging reporter gene mice, we will test the hypothesis that a dynamic pattern of vascular endothelial growth factor (VEGF) receptor 2 and VEGF expression occurs during bone defect healing and that the transcription factor early growth response gene 1 (EGR-1) is an in vivo regulator of these angiogenic transcription pathways. The second aim focuses on bone precursor cell proliferation, an event that typically precedes bone matrix and mineral formation. Current models describe cell proliferation as crucial for de novo bone formation on orthopaedic biomaterials. However, the in vivo timing, magnitude, and specificity of this biological response remain unclear. Using a novel imaging-based proliferation measurement, we propose to test, in an in vivo environment, whether biomaterial surfaces provoke differential proliferative responses in primary human bone precursor cells. Our third aim will provide proof-of-principle that enzymatic bone cell activity can be detected and measured directly in living, intact bone using a small imaging molecule and a clinically relevant imaging paradigm. Together, the proposed studies will serve as model for future experiments which utilize imaging technology in addition to traditional ex vivo methods for the analysis of bone formation pathways. PUBLIC HEALTH RELEVANCE: This interdisciplinary project plans to investigate the biology that controls osteogenesis in living bone under physiological conditions. To begin to establish the relevance of osteogenic transcription, cell proliferation and bone cell activity in vivo, our three aims will introduce and validate transgenic imaging reporter gene mice, imaging-based proliferation measurement and an innovative small imaging molecule strategy, respectively. In addition, we will utilize the transgenic animals to study angiogenic transcription during bone healing and take advantage of the in vivo proliferation measurement to investigate the growth response associated with de novo bone formation.